EP1520011A2 - Sterilisierung von proteinhydrolysat enthaltenden fermentationsausträgen - Google Patents

Sterilisierung von proteinhydrolysat enthaltenden fermentationsausträgen

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Publication number
EP1520011A2
EP1520011A2 EP03735323A EP03735323A EP1520011A2 EP 1520011 A2 EP1520011 A2 EP 1520011A2 EP 03735323 A EP03735323 A EP 03735323A EP 03735323 A EP03735323 A EP 03735323A EP 1520011 A2 EP1520011 A2 EP 1520011A2
Authority
EP
European Patent Office
Prior art keywords
enzyme
fermentation
complex
interest
sources
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP03735323A
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English (en)
French (fr)
Inventor
Mogens WÜMPELMANN
Niels Banke
Soren Michaelsen
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Novozymes AS
Original Assignee
Novozymes AS
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Filing date
Publication date
Application filed by Novozymes AS filed Critical Novozymes AS
Publication of EP1520011A2 publication Critical patent/EP1520011A2/de
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C12N9/2414—Alpha-amylase (3.2.1.1.)
    • C12N9/2417—Alpha-amylase (3.2.1.1.) from microbiological source
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus

Definitions

  • the present invention relates to a method of fermenting an enzyme of interest in a 5 more economical way by adding one or more partially prehydrolysed complex N-sources to the fermentation medium.
  • the media used for fermentative production of valuable compounds on an 0 industrial scale contain normally traditional N-sources such as soy, or corn steep liquor, or yeast extracts.
  • N-sources such as soy, or corn steep liquor, or yeast extracts.
  • the drawbacks by using these traditional N-sources are high viscosity, raw material variation, problematic recovery, formation of coloured substances during heat sterilisation or that the N-source is too costly or used too fast.
  • minimal media may be used, e.g. as s suggested in WO 98/37179, but the drawbacks here are slow outgrowth and low yields.
  • WO 01/05997 describes production of Tetanus Toxin by using a media comprising hydrolyzed soy; the inventors state on page 67 that autoclaving glucose with the rest of the medium is beneficial for seed growth and toxin production.
  • a method for the production of an enzyme of interest, on an industrial scale comprising a) fermentation of a microbial strain producing an enzyme of interest in a fermentation medium comprising one or more partially prehydrolysed complex N-source(s), wherein said partially prehydrolysed N-source(s) are sterilised separately from any other source containing carbohydrates, the prehydrolysis being achieved by addition of an acid and/or a 0 hydrolytic enzyme; and b) recovery of the enzyme of interest from the fermentation broth.
  • microorganism (the microbial strain) according to the invention may be obtained from microorganisms of any genus.
  • the enzyme of interest may be obtained from a bacterial or a fungal source.
  • the enzyme of interest may be obtained from a gram positive bacterium such as a Bacillus strain, e.g., Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus coagulans, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megate um, Bacillus stearothermophilus, Bacillus subtilis, or Bacillus thuringiensis; or a Streptomyces strain, e.g., Streptomyces lividans or Streptomyces murinus; or from a gram negative bacterium, e.g., E. coli or Pseudomonas sp.
  • a Bacillus strain e.g., Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus coagulans, Bac
  • the enzyme of interest may be obtained from a fungal source, e.g. from a yeast strain such as a Candida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia strain, e.g., Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis or Saccharomyces oviformis strain.
  • yeast strain such as a Candida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia strain, e.g., Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Sac
  • the enzyme of interest may be obtained from a filamentous fungal strain such as an Acremonium, Aspergillus, Aureobasidium, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, or Trichoderma strain, in particular the enzyme of interest may be obtained from an Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium
  • ATCC American Type Culture Collection
  • DSM Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH
  • CBS Centraalbureau Voor Schimmelcultures
  • NRRL Northern Regional Research Center
  • the term "obtained from” as used herein in connection with a given source shall mean that the enzyme of interest is produced by the source or by a cell in which a gene from the source has been inserted.
  • the enzyme of interest may be a peptide or an enzyme.
  • a preferred peptide according to this invention contains from 5 to 100 amino acids; preferably from 10 to 80 amino acids; more preferably from 15 to 60 amino acids; even more preferably from 15 to 40 amino acids.
  • the method is applied to enzymes, in particular to hydrolases (class EC 3 according to Enzyme Nomenclature; Recommendations of the Nomenclature Committee of the International Union of Biochemistry).
  • proteases include those of animal, vegetable or microbial origin. Microbial origin is preferred. Chemically modified or protein engineered mutants are included.
  • the protease may be an acid protease, a serine protease or a metallo protease, preferably an alkaline microbial protease or a trypsin-like protease.
  • Cell free culture broth is produced from the culture broth by filtration, centrifugation or similar processes separating insoluables (incl. cells) from the soluables in the broth.
  • alkaline proteases are subtilisins, especially those derived from Bacillus, e.g., subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 and subtilisin 168 (described in WO 89/06279).
  • trypsin-like proteases are trypsin (e.g. of porcine or bovine origin) and the Fusarium protease described in WO 89/06270 and WO 94/25583.
  • useful proteases are the variants described in WO 92/19729, WO
  • Preferred commercially available protease enzymes include ALCALASETM, SAVINASETM, PRIMASETM, DURALASETM, ESPERASETM, RELASETM and KANNASETM (Novozymes A/S), MAXATASETM, MAXACALTM, MAXAPEMTM, PROPERASETM, PURAFECTTM, PURAFECT OXPTM, FN2TM, and FN3TM (Genencor International Inc.).
  • Peptidases An example of a suitable peptidase is FLAVOURZYMETM (Novozymes A/S).
  • Lipases Suitable lipases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful lipases include lipases from Humicola (synonym Thermomyces), e.g. from H. lanuginosa (T. lanuginosus) as described in EP 258 068 and EP 305 216 or from H. insolens as described in WO 96/13580, a Pseudomonas lipase, e.g. from P. alcaligenes or P.
  • pseudoalcaligenes EP 218 272
  • P. cepacia EP 331 376
  • P. stutze GB 1 ,372,034
  • P. fluorescens Pseudomonas sp. strain SD 705 (WO 95/06720 and WO 96/27002)
  • P. wisconsinensis WO 96/12012
  • Bacillus lipase e.g. from B. subtilis (Dartois et al. (1993), Biochemica et Biophysica Acta, 1131 , 253- 360
  • S. stearothermophilus JP 64/744992
  • B. pumilus WO 91/16422).
  • lipase variants such as those described in WO 92/05249, WO 94/01541 , EP 407 225, EP 260 105, WO 95/35381 , WO 96/00292, WO 95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO 97/04079 and WO 97/07202.
  • Preferred commercially available lipase enzymes include LIPOLASETM, LIPOLASE ULTRATM and LIPEXTM (Novozymes A/S).
  • Amylases Suitable amylases ( ⁇ and/or ⁇ ) include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Amylases include, for example, oc-amylases obtained from Bacillus, e.g. a special strain of B. licheniformis, described in more detail in GB 1 ,296,839.
  • amylases examples include the variants described in WO 94/02597, WO 94/18314, WO 96/23873, and WO 97/43424, especially the variants with substitutions in one or more of the following positions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 181 , 188, 190, 197, 202, 208, 209, 243, 264, 304, 305, 391 , 408, and 444.
  • Commercially available amylases are DURAMYLTM, TERMAMYLTM, FUNGAMYLTM,
  • Suitable cellulases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Suitable cellulases include cellulases from the genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, e.g.
  • cellulases are the alkaline or neutral cellulases having colour care benefits.
  • Examples of such cellulases are cellulases described in EP 0 495 257, EP 0 531 372, WO 96/11262, WO 96/29397, WO 98/08940.
  • Other examples are cellulase variants such as those described in WO 94/07998, EP 0 531 315, US 5,457,046, US 5,686,593, US 5,763,254, WO 95/24471 , WO 98/12307 and PCT/DK98/00299.
  • cellulases include CELLUZYMETM, and CAREZYMETM (Novozymes A/S), CLAZINASETM, and PURADAX HATM (Genencor International Inc.), and KAC-500(B)TM (Kao Corporation). Oxidoreductases
  • Oxidoreductases that may be treated according to the invention include peroxidases, and oxidases such as laccases, and catalases.
  • Other preferred hydrolases are carbohydrolases including MANNAWAYTM
  • Novozymes A/S and pectate lyase (e.g. BIOPREPARATION 3000TM (Novozymes A/S)).
  • Other preferred enzymes are transferases, lyases, isomerases, and ligases.
  • suitable complex N-sources are proteins of plant or animal origin, in particular proteins of plant or animal origin containing less than 10 % of carbohydrate; in particular containing less than 5 % of carbohydrate; especially containing less than 3 % of carbohydrate.
  • the amount of prehydrolysed complex N-sources added to the fermentation medium is of at least 5 % (w/w) of the total amount of N-Kjeldahl added to the fermentation medium, in particular of 10-75% (w/w) of the total amount of N-Kjeldahl added to the fermentation medium.
  • Enzymatic prehydrolysis of the complex N-source is preferred, but the invention may also be carried out using other techniques such as acid hydrolysis. Examples of preferred embodiments of prehydrolysis procedures are given.
  • the desired degree of prehydrolysis is preferably achieved by properly adjusting the hydrolysis temperature, the amount of protease and/or peptidase added, the time allowed for the prehydrolysis to occur and by the selection of hydrolytic enzymes used in the prehydrolysis in conjunction with the selection of proper pH intervals for the prehydrolysis to occur with the hydrolytic enzymes chosen.
  • the desired degree of prehydrolysis would depend on several factors: From the perspective of achieving high product titers and thus high volumetric product productivities the use of highly concentrated feed media is potentially advantageous. Thus, adding separately sterilised complex N-sources to the feed medium should be avoided if sufficient amounts of readily utilisable complex N-sources - gradually throughout the fermentation - can be made available from not readily available complex N-sources in the make-up medium present in the fermentor prior to inoculation in order for the biomass formation and/or the product formation to become stimulated.
  • pumpable is used to characterise a suspension of solid particles that rarely forms clumps in pumps, valves and piping systems used - the presence of such clumps altering feed rates by more than 5%.
  • the prehydrolysis is preferably giving rise to breakage of between 10 and 70% of the peptide bonds, more preferably between 15 and 40% of the peptide bonds.
  • the prehydrolysis is preferably giving rise to breakage of between 1 and 20% of the peptide bonds, more preferably between 2 and 10% of the peptide bonds.
  • the enzyme of interest is a self-destructive protease or a peptidase it might be especially advantageous to use as the complex N-source a mixture of highly hydrolysed protein and only slightly hydrolysed protein the preferred degree of prehydrolysis thus stated above for producing such enzymes of interest, for the total amount of complex N-source added, being calculated as:
  • DPH(highly hydr.) is the degree of prehydrolysis of the highly hydrolysed protein
  • DPH(slightly hydr.) is the degree of prehydrolysis of the slightly hydrolysed protein
  • W(highly hydr.) is the weight of highly hydrolysed protein used in the medium
  • W(slightly hydr.) is the weight of slightly hydrolysed protein used in the medium.
  • the present invention may be useful for any fermentation in industrial scale, e.g. for any fermentation having culture media of at least 50 litres, preferably at least 100 litres, more preferably at least 500 litres, even more preferably at least 1000 litres, in particular at least 5000 litres.
  • the microbial strain may be fermented by any method known in the art.
  • the fermentation medium may be a complex medium comprising complex nitrogen and carbon sources.
  • the fermentation may be performed as a batch, a repeated batch, a fed-batch, a repeated fed-batch or a continuous fermentation process.
  • a fed-batch process either none or part of the compounds comprising one or more of the structural and/or catalytic elements is added to the medium before the start of the fermentation and either all or the remaining part, respectively, of the compounds comprising one or more of the structural and/or catalytic elements is fed during the fermentation process.
  • the compounds which are selected for feeding can be fed together or separate from each other to the fermentation process.
  • the complete start medium is additionally fed during fermentation.
  • the start medium can be fed together with or separate from the structural element feed(s).
  • part of the fermentation broth comprising the biomass is removed at regular time intervals, whereas in a continuous process, the removal of part of the fermentation broth occurs continuously.
  • the fermentation process is thereby replenished with a portion of fresh medium corresponding to the amount of withdrawn fermentation broth.
  • a fed-batch, a repeated fed-batch process or a continuous fermentation process is preferred.
  • a further aspect of the invention concerns the downstream processing of the fermentation broth.
  • the enzyme of interest may be recovered from the fermentation broth, using standard technology developed for the enzyme of interest.
  • the degree of hydrolysis was determined as described in Example 4 assuming a dry matter content in potato protein of 93% and a protein content in potato protein as % of dry matter of 80%.
  • the degree of hydrolysis was determined as described in Example 4 assuming a dry matter content in potato protein of 93% and a protein content in potato protein as % of dry matter of 80%.
  • the degree of hydrolysis was determined as described in Example 4 assuming a dry matter content in potato protein of 93% and a protein content in potato protein as % of dry matter of 80%.
  • the mixture was centrifuged until the supernatant was clear. The supernatant was then appropriately diluted with deionised water (to V1 ml).
  • the average OD must be between OD measured for blind and standard; otherwise the dilution was changed accordingly.
  • sample the average OD(340 nm) value measured for the sample ODav.
  • standard the average OD(340 nm) value measured for the serine standard ODav.
  • blind the average OD(340 nm) value measured for the blind
  • V1 (ml) dilution volume in mL
  • W1 (mg) sample in mg
  • constant chosen for potato protein D 9.1 , constant chosen for potato protein
  • the OPA value is thus reflecting the percentage of peptide bonds hydrolysed within the sample analysed.
  • Example 5 Strains The protease strain used in Example 6 (Af50-34) and further used in Example 7 and 8 was an isolate of NCIB 10309 and genetically modified as described in EP 0506780 B1.
  • a spontaneous rifampicin-resistant mutant was isolated which contained a substitution of amino acid number 478 in the RpoB protein from alanine to valine, resulting in strain SJ4671 riflO disclosed in the copending Danish patent application PA 2001 01972.
  • the fermentation was carried out in 2 liter fermentors equipped with 4 baffles at agitation and aeration rates sufficient to maintain a dissolved oxygen concentration at or above 20% of saturation throughout.
  • the aeration did not at any time exceed 2 l/l/min.
  • the temperature was maintained at 37°C.
  • Antifoam oil - in amounts sufficient to prevent foaming becoming uncontrollable - was added initially to the make-up and the feed medium. pH was maintained between 8.0 and 7.7 by addition of 15% H3PO4 and/or 10% NH3 in water.
  • Feeding medium was initiated at time 0.1 h from inoculation and was maintained at the following rates:
  • Nicotinic acid 0.03 g
  • the fermentation was sampled at 49 h and at 71 h from inoculation and samples analysed for protease activity according to Example 11.
  • the fermentation was carried out in 2 liter fermentors equipped with 4 baffles at agitation and aeration rates sufficient to maintain a dissolved oxygen concentration at or above 20% of saturation throughout.
  • the aeration did not at any time exceed 2 l/l/min.
  • the temperature was maintained at 37°C.
  • Antifoam oil - in amounts sufficient to prevent foaming becoming uncontrollable - was added initially to the make-up and the feed medium. pH was maintained between 7.5 and 7.0 by addition of 15% H3PO4 and/or 10% NH3 in water.
  • Feeding medium was initiated at time 0.1 h from inoculation and was maintained at the following rates:
  • Example 10 Fermentatiom with the SJ 5262 strain; unhydrolysed potato protein in the make-up medium
  • This fermentation was carried out exactly as the fermentation described in Example 9 except that unhydrolysed potato protein was used in the make-up medium in amounts equivalent to the amount of protein hydrolysate used in Example 9 when based on dry matter derived from potato protein present in the hydrolysate/unhydrolysed protein (15 g/l potato protein).
  • the protease enzyme titers (Example 7 and 8) were measured by methods known within the art based on measuring the enzyme activities present in the culture broth samples, e.g., the method for protease activity analysis described in WO 89/06279 (p. 29-31) may be used.
  • the alpha-amylase enzyme titers (Example 9 and 10) were measured by methods known within the art based on measuring the enzyme activities present in the culture broth samples, e.g., the method for alpha-amylase activity analysis described in WO 95/26397 (p. 9-10) may be used.
  • Potato protein hydrolysate; OPA 2.9 in feed (Example 7): relative titer at 49/71 h: 139/130
  • Potato protein hydrolysate; OPA 51 in feed (Example 8): relative titer at 49/71 h: 100/68 (All titers relative to yield at 49 h reached in Example 8) SJ 5262/alpha-amylase:
  • Potato protein hydrolysate; OPA 19.5 in make-up (Example 9): relative titer at 95/116 h: 111/130
  • Unhydrolysed potato protein (Example 10): relative titer at 95/116 h: 100/117

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EP03735323A 2002-07-01 2003-06-26 Sterilisierung von proteinhydrolysat enthaltenden fermentationsausträgen Withdrawn EP1520011A2 (de)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
DK200201021 2002-07-01
DKPA200201021 2002-07-01
DKPA200201838 2002-11-28
DK200201838 2002-11-28
PCT/DK2003/000441 WO2004003216A2 (en) 2002-07-01 2003-06-26 Sterilization of a fermentation medium comprising hydrolysed n-source

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EP1520011A2 true EP1520011A2 (de) 2005-04-06

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EP (1) EP1520011A2 (de)
CN (1) CN1665924A (de)
AU (1) AU2003236810A1 (de)
WO (1) WO2004003216A2 (de)

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